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Impaired Activating Receptor Expression Pattern in Natural Killer Cells from Patients with Multiple Myeloma

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Impaired Activating Receptor Expression Pattern in Natural Killer Cells from Patients with Multiple Myeloma Letters to the Editor 732 specificity for the CD20 molecule for potential use in adoptive by IMF SC 110212, University of Muenster to CS. MP and CS immunotherapy of B-cell malignancies. We show that cTCR þ contributed equally. CD8 þ NKT cells can be expanded in large quantities and are M Pieper1, C Scheffold1, S Duwe2, C Rossig2, G Bisping1, highly effective in lysing CD20 molecule expressing Daudi and 1 3 2 1 1 Raji tumor cell lines, as well as freshly isolated malignant B M Stelljes , TF Tedder , H Jurgens , WE Berdel and J Kienast 1Department of Medicine/Hematology and Oncology, lymphocytes from B-CLL patients. In competitive targeting University of Muenster, Muenster, Germany; studies, we were able to dissect the major pathways of tumor 2 þ Department of Pediatric Hematology and Oncology, cell recognition and found that endowing CD8 NKT cells with University of Muenster, Muenster, Germany and CD20z chimeric receptors resulted in a significant increase of 3Department of Immunology, Duke University, cytotoxic activity against CD20 þ Daudi targets as compared to Durham, NC, USA nontransduced effectors. E-mail: [email protected] We also demonstrate that chimeric receptor mediated cytotoxicity is dependent on the expression level of the target antigen. Tumor targets expressing high levels of CD20 molecule (Daudi) were lysed more efficiently than low level expressing References targets (Raji). Of importance, cytotoxicity of ex vivo expanded cTCR þ CD8 þ NKT cells was more potent as compared to 1 Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and ex vivo expanded cTCR þ CD8 þ T cells. We believe these targeting of cytotoxic lymphocytes through chimeric single chains differences in cytotoxic activity to be a result of cultured consisting of antibody-binding domains and the gamma or zeta þ subunits of the immunoglobulin and T-cell receptors. Proc Natl CD8 T cells being sensitized for activation induced cell Acad Sci USA 1993; 90: 720–724. death (AICD) following repetitive stimulation with IL-2. In 2 Sadelain M, Riviere I, Brentjens R. Targeting tumours with addition, functional cTCR signaling was documented by genetically enhanced T lymphocytes. Nat Rev Cancer 2003; 3: detection of high levels of the Th-1 cytokine IFN-g secreted by 35–45. cTCR þ CD8 þ NKT cells upon CD20 antigen target recognition. 3 Brocker T, Karjalainen K. Signals through T cell receptor-zeta chain Of interest, blocking the adhesion molecules LFA-1/ICAM-1 had alone are insufficient to prime resting T lymphocytes. J Exp Med 1995; 181: 1653–1659. no major influence on chimeric T-cell receptor mediated þ 4 Ortaldo JR, Winkler-Pickett RT, Yagita H, Young HA. Comparative cytotoxicity of CD8 NKT cells. studies of CD3- and CD3+ CD56+ cells: examination of morphol- Our results suggest that cellular immunotherapy using human ogy, functions, T cell receptor rearrangement, and pore-forming ex vivo expanded CD8 þ NKT cells expressing CD20 antigen- protin-expression. Cell Immunol 1991; 136: 486–495. specific cTCR could add to standard treatment regimens for 5 Schmidt-Wolf IG, Lefterova P, Mehta BA, Fernandez LP, Huhn D, CD20 þ B-cell malignancies, for example to overcome recurrent Blume KG et al. Phenotypic characterization and identification of effector cells involved in tumor cell recognition of cytokine-induced or refractory disease, with the advantage of minor additional killer cells. Exp Hematol 1993; 21: 1673–1679. toxicity. However, further studies are needed to determine the 6 Verneris MR, Baker J, Edinger M, Negrin RS. Studies of ex vivo þ þ optimal use of genetically engineered cTCR CD8 NKT cells. activated and expanded CD8+ NK-T cells in humans and mice. Finally, the proposed immunotherapeutic strategy may also be J Clin Immunol 2002; 22: 131–136. applicable to other neoplastic diseases by modification of the 7 Scheffold C, Kornacker M, Scheffold YC, Contag CH, Negrin RS. candidate gene. Visualization of effective tumor targeting by CD8+ natural killer T cells redirected with bispecific antibody F(ab’)(2)HER2xCD3. Cancer Res 2002; 62: 5785–5791. 8 Kansas GS, Tedder TF. Transmembrane signals generated through Acknowledgements MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent and independent adhesion in human B cells We are grateful for technical assistance by Colette Huenefeld and through a tyrosine kinase-dependent pathway. J Immunol 1991; statistical analysis by Dr Andreas Grote. This work was supported 147: 4094–4102. Impaired activating receptor expression pattern in natural killer cells from patients with multiple myeloma Leukemia (2006) 20, 732–733. doi:10.1038/sj.leu.2404096; tested the hypothesis of an additional mechanism involving the published online 26 January 2006 effector cell, that is, NK, as previously described in leukemic patients.3 We analyzed peripheral NK from six MM patients at diag- Multiple myeloma (MM) is a hematological malignancy charac- nosis. The activating receptor expression was checked on terized by accumulation in the bone marrow of plasma cells CD19ÀCD3ÀCD56 þ cells by flow cytometry using mAbs against that secrete a monoclonal immunoglobulin (Ig). The implication NKp30, NKp44, NKp46, NKG2D, CD16 and 2B4/CD244 (for of natural killer cells (NK) in antitumor immunity against MM patient descriptions, see Supplementary Information). is suggested by plasmocyte susceptibility to NK lysis.1 NK In MM patients, the percentage of NKp30-expressing NK efficiently kill early-stage MM cells by a natural cytotoxicity (69%718) and the mean fluorescence intensity (MFI ¼ 42731) receptor (NCR)- and NKG2D-dependent pathway.2 In contrast, did not statistically differ (P ¼ 0.28, Mann–Whitney non-para- late-stage MM cells are protected from NK lysis by a high metric test; Figure 1) from 23 healthy donors (86%711 positive expression of HLA class I molecules.2 In addition to this cells, MFI ¼ 56737). In MM patients, the percentage of NKp46- immune evasion mechanism involving the target cell,2 we expressing NK (80%715) and the MFI (62735) did not Leukemia Letters to the Editor 733 NKp46 NKp30 CD56 250 150 500 200 400 100 150 300 100 50 200 50 100 0 0 0 NKG2D 2B4 CD56 ** 150 600 * 500 500 400 100 400 300 300 200 50 200 100 100 0 0 0 Figure 1 Partially defective triggering receptor pattern in MM NK. NK were identified as CD19ÀCD3ÀCD56 þ cells, and further analyzed for NCR, NKG2D, CD16 and 2B4 expression. Each plot corresponds to data from one patient, together with the mean MFI7standard deviation. nP ¼ 0.001, nnP ¼ 0.02 (bidirectional Mann–Whitney non-parametric test). statistically differ (P ¼ 0.18) from healthy donors (85%78 Acknowledgements positive cells, MFI ¼ 86758). In MM patients, we did not detect NKp44 expression on resting NK, as the expression of this This work was supported by Groupement entreprise franc¸aise lutte receptor is restricted to activated NK.4 All NK from MM patients cancer, association pour la recherche contre le cancer, Fe´de´ration or healthy donors expressed NKG2D. The MFI of NKG2D was nationale des centres de lutte contre le cancer, Fondation pour la lower in MM patients than in healthy donors, but the difference Recherche Me´dicale. 7 7 did not reach statistical difference (65 35 vs 83 13, P ¼ 0.13). 1 1 1,3 1,2,3 The 2B4/CD244 receptor was expressed on all NK, both in MM C Fauriat , F Mallet , D Olive and RT costello 1Laboratoire d’Immunologie des Tumeurs INSERM U599, and healthy donors. Nonetheless, 2B4/CD244 MFI in MM Institut Paoli-Calmettes, Marseille, France; patient NK was drastically lower than in healthy donors 2 ´ ´ ˆ 7 7 Departement d’Hematologie, Hopital Nord, Marseille, (129 61 vs 304 119, P ¼ 0.001). NK from MM patients all France and expressed CD16, but the MFI was reduced in MM patients 3Universite´ de la Me´diterrane´e, Marseille, France compared to healthy donors NK (117765 vs 2307123, E-mail: [email protected] P ¼ 0.021). As an invariant control, no significant differences were observed regarding the expression of CD56 on NK in References healthy donors vs MM patients. We conclude to a normal expression of NCR and NKG2D in 1 Frohn C, Hoppner M, Schlenke P, Kirchner H, Koritke P, Luhm J. 2 MM patients, in agreement with data from Carbone et al. Anti-myeloma activity of natural killer lymphocytes. Br J Haematol Nonetheless, the coreceptor 2B4/CD244 and the low-affinity Ig- 2002; 119: 660–664. Fc receptor CD16 displayed significantly weaker expression in 2 Carbone E, Neri P, Mesuraca M, Fulciniti MT, Otsuki T, Pende D comparison with healthy donors. Continuous exposure to the et al. HLA class I, NKG2D, and natural cytotoxicity receptors circulating monoclonal Ig may explain CD16 downmodulation. regulate multiple myeloma cell recognition by natural killer cells. Blood 2005; 105: 251–258. This downregulation might more specifically impair the anti- 3 Costello RT, Sivori S, Marcenaro E, Lafage-Pochitaloff M, Mozzi- body-dependent cytotoxicity. We have demonstrated that MM conacci MJ, Reviron D et al. Defective expression plasmocytes express CD48, the ligand for 2B4/CD244 (shown in and function of natural killer cell-triggering receptors in Supplementary Information). As a consequence of 2B4/CD244 patients with acute myeloid leukemia. Blood 2002; 99: downregulation, NK might lack the NCR costimulation provided 3661–3667. by CD48-expressing plasmocytes. Defective CD48-2B4/CD244 4 Vitale M, Bottino C, Sivori S, Sanseverino L, Castriconi R, Marcenaro E et al. NKp44, a novel triggering surface molecule interaction is a novel mechanism that may further favor, in specifically expressed by activated natural killer cells, is involved addition to the upregulation of HLA class I, escape of MM in non-major histocompatibility complex-restricted tumor cell lysis. plasmocytes to innate immunity.2 J Exp Med 1998; 187: 2065–2072.
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